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    16 June 2020, Volume 53 Issue 12
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Genome-Wide Identification and Expression Analysis of PIN Genes Family in Wheat
    LIU PeiXun,WAN HongShen,ZHENG JianMin,LUO JiangTao,PU ZongJun
    Scientia Agricultura Sinica. 2020, 53(12):  2321-2330.  doi:10.3864/j.issn.0578-1752.2020.12.001
    Abstract ( 1038 )   HTML ( 139 )   PDF (2968KB) ( 907 )   Save
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    【Objective】 The puroindoline (PIN) gene is a family specific to plants and plays an important role in controlling the grain hardness in wheat. In silico identification and expression analysis of PIN family genes in Triticum aestivum on whole genome level lay the foundation for elucidation the biological function of wheat PIN family. 【Method】 The known sequences of wheat PIN proteins and barley hordoindoline (HIN) proteins were used to query the newly released wheat peptides dataset of Chinese spring variety with HMM and BLASTP profiles. UniProt, URGI, PFAM, CDD, expVIP and other databases were used for bioinformatics analysis with Clustal X, MEGA 7.0, ExPASy, MEME, GSDS, TBtools, GraphPad Prism5 and other programs. The expression profiles of TaPIN genes in different wheat harness seeds were validated with quantitative real-time polymerase chain reaction (qRT-PCR). 【Result】 A total of 19 TaPINs were identified in wheat genome, which were clustered in homologous groups of chromosome 1, 5 and 7. These TaPIN proteins contained 148 to 327 amino acids, as their relative molecular weight varied from 16.39 to 37.19 kD and the isoelectric points ranged from 6.35 to 9.34. Phylogenetic and conserved domain analyses showed that the 19 TaPIN proteins were divided into A and B categories, respectively. Most TaPIN genes harbored only one exon, and there were many cis-acting regulatory elements involved in stress responsiveness and seed-specific regulation. RNA-Seq showed that this gene family expressed largely in wheat grain and hardly expressed in other tissues. The qRT-PCR results indicated that the relative expression level among TaPIN genes were significantly changed. TaPIN9 and TaPIN10 were highly expressed. The expression levels of TaPIN9 and TaPIN10, as well as its expression ratio, were up-regulated with the decrease of wheat grain hardness, while TaPIN16 and TaPIN6 showed an opposite trend. 【Conclusion】 Pina and Pinb genes were the main factors regulating grain hardness in wheat. It is speculated that other members of this gene family have similar functions, but have little influence on grain hardness because of low expression level. According to the evolutionary relationship of this gene, Aegilops tauschii is most closely related to wheat, followed by oats, rye and barley.

    Expression Analysis of the Chlorophyll Biosynthesis Structural Genes in Green and White Foxtail Millet [Setaria italica (L.) Beauv]
    ZHANG Bin,LI Meng,LIU Jing,WANG JunJie,HOU SiYu,LI HongYing,HAN YuanHuai
    Scientia Agricultura Sinica. 2020, 53(12):  2331-2339.  doi:10.3864/j.issn.0578-1752.2020.12.002
    Abstract ( 507 )   HTML ( 47 )   PDF (2690KB) ( 483 )   Save
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    【Objective】 The phenotypic differences of millet color in foxtail millet is caused by the different kinds of pigment accumulation in grains. The phenotype of millet color, the indicators from color system, the chlorophyll contents, the ultra-structure of inner grains, and the expression profile of chlorophyll biosynthesis related genes were compared and analyzed between the green and white millet varieties. Our main objectives are to find the key gene causing the millet color difference between green and white millet, and to explore the mechanism for the formation of green millet color. 【Method】 In this study, the millet color indicators (L*, a*, b*)were determined using the color system. The Chla, Chlb, and Chl contents in grains were measured by spectrophotometer separately, and the inner ultra-structure including the size and numbers of the starch and chromoplast in the foxtail millet grains were observed and analyzed at mid stage of grouting by transmission electron microscope. Meanwhile, the expression patterns of 18 genes distributing in the upstream and downstream of the chlorophyll biosynthesis were analyzed among three varieties with different millet color by qRT-PCR. The SiCAO gene was separately cloned from three varieties with the DNA of seedling leaves as templates, and then were compared with each other. 【Result】 Hunted millet of Daqinggu and Lumiqinggu were green colors, and the a* color indicators of them were lower than Niumaobai with white millet color. The Chla, Chlb and Chlcontains in the grains of green varieties were all significantly higher than white variety, and only 0.006 mg·g-1 Chlb were detected in white variety. The results of qRT-PCR showed that the expression of SiCAO gene couldn’t be detected in white variety, but it had high expression levels in two green varieties. The significant different expression level of SiCAO was one of the important reasons leading to the color difference between two millet color millet varieties. The SiCAO gene with an open reading frame of 1626 bp was separately cloned from three varieties, and their sequences showed differences at 171, 184, 195, 286 and 318 amino acid sites. Simultaneously, ultra-structural observations indicated that the endosperm layer of the grain is mainly consisting of the single-amyloplast. Compared with the white variety, the starch in green varieties were bigger and darker, and surrounded by more chromoplasts and spherosomes, and these differences might affect the pigments accumulations in grains. 【Conclusion】 SiCAO, which is over-expressed in green varieties but not expressed in white variety, is one of the important reasons that causing different amount of chlorophyll contents accumulation leading to green and white millet colors.

    Development of an Efficient Editing System in Arabidopsis by CRISPR-Cas9
    ZHANG Cheng,HE MingLiang,WANG Wei,XU FangSen
    Scientia Agricultura Sinica. 2020, 53(12):  2340-2348.  doi:10.3864/j.issn.0578-1752.2020.12.003
    Abstract ( 691 )   HTML ( 41 )   PDF (3127KB) ( 674 )   Save
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    【Background】 In recent years, the CRISPR-Cas9 system has been developed to an efficient site-directed gene- editing technology in a variety of plants. It can provide a rapid method for studying gene function. However, the editing efficiency of some CRISPR-Cas9 vectors is very low. 【Objective】 In this study, an efficient CRISPR-Cas9 system, with a red fluorescent protein screening marker and a RIBOSOMAL PROTEIN S5 A (RPS5A) promoter, was constructed to edit the xyloglucan endotransglucosylase/ hydrolase TOUCH4 (TCH4) gene in Arabidopsis thaliana. 【Method】 The CRISPR vector pRSE-WH was constructed from the pKSE401 vector by replacing the 35S promoter with a RPS5A promoter, which maintains high constitutive expression at all developmental stages from the egg cell, and the hygromycin resistance gene with DsRed2. The sgRNA of TCH4 was designed using CRISPR-P 2.0 (http://crispr.hzau.edu.cn). After excluding non-specific target sites by sequence comparisons with the Arabidopsis thaliana genome database, two target sites were selected. The sgRNA oligonucleotides with junctions were chemically synthesized. After annealing, two expression vectors (TCR1 and TCR2, corresponding to the two target sites) were constructed by linking the sgRNA with junctions to the plasmid pRSE-WH respectively. The vectors of TCR1 and TCR2 were transferred into wild-type Col-0 by Agrobacterium-mediated floral-dip transformation. In the T1 generation, the positive transgenic plants were identified using red fluorescent protein as a marker, and the target sites were detected using PCR combined with Sanger-sequencing to confirm whether it was mutated as expected. The mutations were determined from the peak map of the sequencing results, and the mutation pattern and genotypes were further analyzed. 【Result】 A high efficiency CRISPR vector pRSE-WH for gene editing in Arabidopsis was constructed. The TCR1 and TCR2 expression vectors successfully enabled gene-specific editing of TCH4. In the editing of the TCH4 gene, the mutation ratios using TCR1 and TCR2 were 80%, 100% respectively, and the total editing efficiency was 86%. The mutations in T1 plants were decoded according to the sequencing results and included homozygous, heterozygous and biallelic mutations. Among 59 mutated T1 plants, 8 plants (13.56%) were not edited, 9 plants (15.25%) were homozygous, 40 plants (67.80%) were biallelic and 2 plants (3.39%) were heterozygous. The homozygous and biallelic seeds without red fluorescence of T1 generation were selected for breeding, and sequenced the plants of T2 generation. The results showed that mutations of the T1 generation were successfully inherited to T2 generation, which were Cas9-free. 【Conclusion】 PRSE-WH demonstrated high-efficiency gene editing in Arabidopsis thaliana. It was very easy to obtain Cas9-free and stably inherited mutants in the T3 generation by screening seeds with red fluorescence marker.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Effects of Plastic Film Mulching on Yield and Water Use of Maize in the Loess Plateau
    XU JiaXing,FENG YongTao,YE YuLian,ZHANG RunZe,HU ChangLu,LEI Tong,ZHANG ShuLan
    Scientia Agricultura Sinica. 2020, 53(12):  2349-2359.  doi:10.3864/j.issn.0578-1752.2020.12.004
    Abstract ( 473 )   HTML ( 37 )   PDF (497KB) ( 398 )   Save
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    【Objective】 Present study comprehensively evaluated effectiveness of the yield and water use under plastic film mulching for maize production, so as to provide a theoretical basis for the rational utilization of plastic film mulching in the Loess Plateau area. 【Method】 The published data about plastic film mulching of maize were collected in the Loess Plateau from 2000 to 2017, using CNKI, Web of Science and EI Compondex, to analyze the effects of plastic film mulching on maize yield, water use and water use efficiency. 【Result】 The results showed that, the average yield of 50% film mulching was 9 348 kg·hm-2, which was 33% higher than the control of 7 059 kg·hm-2; The average yield of 100% film mulching was 9 191 kg·hm-2, which was 56% higher than the control of 5 910 kg·hm-2; Compared with no film mulching, both 100% film mulching and 50% film mulching significantly increased the yield of maize. However, the yield of maize under 100% film mulching and 50% film mulching was similar. In addition, the increase of maize yield was significantly and positively related with the increase of altitude, which was higher than 900 m under 100% film mulching and 627 m under 50% film mulching, respectively. This was possibly ascribed to the increase of soil temperature under film mulch. The average evapotranspiration (ET) of 50% film mulching was 385 mm, which was 1% lower than the control of 389 mm; The average ET of 100% film mulching was 366 mm, which was 2.5% higher than the control of 357 mm, the ET under 50% film mulching and the control was similar, but 100% film mulching significantly increased evapotranspiration relative to the control, while there was no significant difference between the two mulching treatments. The average water use efficiency (WUE) of 50% film mulching was 17.3 kg·hm-2·mm-1, which was 31% higher than the control of 13.2 kg·hm-2·mm-1; The average WUE of 100% film mulching was 17.5 kg·hm-2·mm-1, which was 50% higher than the control of 11.7 kg·hm-2·mm-1; Compared with the control, both 100% film mulching and 50% film mulching significantly increased the WUE of maize. However, the WUE of maize under 100% film mulching and 50% film mulching was similar. The significant increase of maize yield and WUE by film mulching was related to the decrease of soil evaporation by 57 mm and the increase of potential transpiration efficiency by 11.2 kg·hm-2·mm-1. 【Conclusion】 At the regional scale, 50% film mulching and 100% film mulching had equivalent effects on maize yield, evapotranspiration and water use efficiency. Considering the environmental pollution and economic cost caused by film mulching, 50% film mulching was recommended for maize cultivation in the Loess Plateau.

    Surface Defect Detection of Navel Orange Based on Region Adaptive Brightness Correction Algorithm
    ZHANG Ming,WANG Teng,LI Peng,DENG Lie,ZHENG YongQiang,YI ShiLai,LÜ Qiang,SUN RongRong
    Scientia Agricultura Sinica. 2020, 53(12):  2360-2370.  doi:10.3864/j.issn.0578-1752.2020.12.005
    Abstract ( 349 )   HTML ( 20 )   PDF (2665KB) ( 425 )   Save
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    【Objective】 Because of uneven lightness distribution on navel orange surface due to its geometric shape of sphere to oval, it is difficult to segment directly the defect area from the orange image by single threshold method. In order to solve this problem, a novel detection method of the surface defects of navel orange based on the region brightness adaptive correction algorithm was proposed in this study. 【Method】 The Newhall navel orange with high economic value was selected as the research object, and its original image was collected using self-developed image acquisition system. During this experiment, it was found that the gray level of R-B fused component image had obvious bimodal distribution, and the color contrast between the background and the foreground of the original image was relatively large. Therefore, the background was removed using the mask generated by R-B fused component image and the single threshold method. The R-B target image was extracted with the background removed, which could effectively remove the background without damaging the navel orange surface. Then, the brightness information of the target image was calculated based on the region adaptive brightness correction algorithm proposed in this work, and the brightness information was estimated according to the set of brighter pixels in the neighborhood of the points on the target image. In this study, the neighborhood window size was set to w×w, and the value of w was set to 13 by experiment comparison. The pixels in the neighborhood were moved on the target image pixel by pixel, and a new matrix was generated. The new matrix was sorted in ascending order in the column, then the last row was removed to exclude the influence of white noise, and the last five rows were selected and the matrix was obtained by means of column operation. The matrix was inversely transformed and stored. Finally, the matrix image with the same size of the target image, that was, the surface brightness information of the target image was obtained. Based on the obtained luminance information, the R-B fused component image after background removal was corrected by luminance homogenization. After brightness correction, the gray contrast between the defect area and the normal tissue area of the image was relatively large. The single threshold method (segmentation threshold T=194) could be used to extract the surface defect directly from the brightness corrected navel orange image. Finally, the area filter was applied to the binary image after surface defect segmentation to remove the spurious points and noise. 【Result】 The bimodal method could effectively remove the background of the image and preserve the surface information of the target navel orange. All the sample images were also corrected and inspected by using other brightness correction algorithms, including histogram equalization algorithm, Retinex theory algorithm and illumination-reflection theory algorithm. The experimental results showed that the algorithm proposed in this study could correct both the middle and the edge of the normal region of the image, which were to be highlighted and the fluctuation range of the gray scale difference was small, while the defect regions still exhibited low gray level. The average processing time of each image was 0.29 seconds in proposed algorithm. Compared with the other three algorithms, the average processing time of each image could be reduced 0.27 s, 0.14 s, and 1.45 s by proposed algorithm. Finally, the algorithm proposed in this study combined with single threshold method, and it was used to detect the defects of 356 Newhall navel orange samples. Based on this algorithm, 8 kinds of common navel orange surface defects (e.g., canker, thrips, scale infestation, insect injury, blackspot, wind damage, anthracnose, dehiscent) were detected, and the defect recognition rate reached 95.8%. The correct defect recognition rate was improved by 2.6%-8.2% which compared with other three algorithms. 【Conclusion】 The proposed algorithm effectively solved the problem of defect segmentation caused by uneven brightness distribution on the surface of spherical fruits, provided technical support for online accurate classification of navel orange fruits, and provided a new idea for rapid detection of surface defects for other fruits.

    PLANT PROTECTION
    Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31
    QI Yue,LÜ JunYuan,ZHANG Yue,WEI Jie,ZHANG Na,YANG WenXiang,LIU DaQun
    Scientia Agricultura Sinica. 2020, 53(12):  2371-2384.  doi:10.3864/j.issn.0578-1752.2020.12.006
    Abstract ( 396 )   HTML ( 23 )   PDF (3446KB) ( 263 )   Save
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    【Objective】 Wheat leaf rust caused by Puccinia triticina (Pt) is one of the main diseases affecting wheat production. In the process of interaction between wheat and Pt, the Pt will secrete some effector proteins to the host cells to regulate the host and play a virulent function. The objective of this study is to carry out the research on Pt effector proteins, explore the pathogenic mechanism of Pt, and to provide a basis for the continuous prevention and control of diseases. 【Method】 The cDNA of Pt 13-5-72 interacting with susceptible variety Thatcher was used as template to amplify effector protein Pt18906. Sequence features of Pt18906 were analyzed using SignalP 4.1, TargetP 1.1, TMHMM 2.0 and EffectorP 2.0 software. The online software Swiss-Model was used to predict the tertiary structure of Pt18906. The secondary structure of Pt18906 was predicted by online software SOPMA. The expression pattern of Pt18906 was analyzed by RT-qPCR. The verification that Pt18906 inhibited Bax and INF1-induced programmed cell death (PCD) was conducted using Nicotiana benthamiana heterogeneous expression system. The secretion function of Pt18906 signal peptide was verified by the yeast system. The deletion mutation of Pt18906 was used to identify the virulence function motif. Subcellular localization of Pt18906 was analyzed using transient expression of Pt18906-GFP in N. benthamiana cells. Transient expression technology was used to carry out the avirulent function of Pt18906 on a full set of near-isogenic lines that did not contain disease resistance genes and contained different disease resistance genes with Thatcher as the background. The transient transformation of wheat mediated by the TTSS (Type III secretion system) was used to analyze the activity of regulating the host defense response of Pt18906. 【Result】 A Pt effector Pt18906, significantly upregulated during the interaction between Pt and Thatcher, was obtained from the transcriptome library of interaction between Pt and Thatcher. The full-length ORF sequence with 672 bp was amplified in the cDNA of wheat leaves inoculated by Pt strain 13-5-72. The effector protein lacks known functional domains and motifs. Its working environment is alkaline. Pt18906 could inhibit the PCD induced by Bax from mice and INF1 from Oomycetes by transient expression in N. benthamiana cells, which indicates that the effector protein has virulent function. By constructing deletion mutants, it is clear that the 28-47 amino acids have an important role in its virulent function. The effector protein is located in the nucleus and cytoplasm, which indicates that it acts within the cells. Pt18906 could cause allergic necrosis in the disease-resistant varieties of single gene lines TcLr27+31 and TcLr42, indicating that the effector protein is avirulent. Pt18906 could cause the accumulation of reactive oxygen species (ROS) and callose in TcLr27+31 through transiently expressing using a bacterial type III secretion system. The callose increased gradually with the injection time, and ROS reached the highest level at 10 min after injection. 【Conclusion】 The main functional domain of Pt18906 is 28-47 amino acids. Pt18906 can trigger the two-layer defense reaction accompanied with the accumulation of callose in wheat TcLr27+31 and oxidative burst.

    Effect of Nozzle Type on the Spray Performance of Plant Protection Unmanned Aerial Vehicle (UAV)
    GONG ChangWei,MA Yu,YANG Rui,RUAN YanWei,WANG XueGui,LIU Yue
    Scientia Agricultura Sinica. 2020, 53(12):  2385-2398.  doi:10.3864/j.issn.0578-1752.2020.12.007
    Abstract ( 453 )   HTML ( 21 )   PDF (1803KB) ( 430 )   Save
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    【Objective】 Plant protection unmanned aerial vehicle (UAV) has the characteristics of high spray efficiency, good applicability, small crop damage and high safety to the operator. However, drift seriously restricts its popularization and application. As the core component, the nozzle is a key factor affecting droplet drift. The objective of this study is to clarify the atomization performance of different types of nozzles and their effect on spray drift, and to provide theoretical basis for selecting suitable nozzles. 【Method】 In this paper, 20 kinds of common fan-shaped, air suction and conical nozzles were selected, and the distribution span, volume diameter (D50) and percentage of the total volume of fog particles smaller than 150 μm (ΦVol<150 μm) of different types of nozzles were detected by laser particle size analyzer. In an open wind tunnel, the flow rate of different nozzles at 0.3 MPa was firstly measured, then the influence of different types of nozzles on spray drift and particle size characteristics of deposition droplet was evaluated by photo paper and mylar card method. 【Result】 The atomization performance of different nozzles was measured under 0.3 MPa spray pressure. It showed that in common fan-shaped nozzle such as F110-01, F110-015, F110-02 and F110-03, the distribution span and D50 increased significantly with the increase of the model, while ΦVol<150 μm decreased significantly. The air fan nozzle from AFC-01 to AFC-05 and the conical nozzle from HCC80-0075 to HCC80-025 had the same rule. The ΦVol<150 μm of air fan nozzle with the same aperture was smaller than that of fan-shaped nozzle and conical nozzle, while the distribution span and D50 were larger than those of fan-shaped nozzle and conical nozzle. The distribution span and D50 of AFC-01 and IDK120-015 were significantly smaller than those of other types of air fan nozzle. The ΦVol<150 μm of IDK120-015 was significantly lower than that of HCC80-02, F110-015 and F110-03, while the distribution span and D50 were significantly higher than those of HCC80-02 and F110-015. There was no significant difference between the flow of HCC80-02 and IDK120-015, F110-015, which were all significantly lower than that of F110-03. Furthermore, the particle size characteristics and drift amount of drift deposition droplets on the ground were evaluated by using mylar card and photo paper. The effects of nozzle type and drift distance on the D50 and distribution span of drift deposition droplets were extremely significant. The D50 and distribution span of drift distance 3 m were significantly lower than those of 1 m and 2 m. The trend of predicted drift amount calculated by Depositscan software was consistent with the measured drift amount, both of which were HCC80-02>F110-015>F110-03>IDK120-015. After the calculation of anti-drift effect of different nozzles, IDK120-015 had the best anti-drift effect (72.02%), F110-03 was the second, and HCC80-02 was the worst. 【Conclusion】 It is a feasible method to evaluate the drift of droplets through mylar card and photo paper to collect the ground drift. The reasonable selection of nozzle can significantly reduce the percentage of small droplets and expand the relative droplet size, and result in the decreased droplets drift during the operation of UAV.

    Resistance Level and Mechanism of Descurainia sophia to Florasulam in Wheat Field of Shandong Province
    GAO XingXiang,ZHANG YueLi,LI Mei,LI Jian,FANG Feng
    Scientia Agricultura Sinica. 2020, 53(12):  2399-2409.  doi:10.3864/j.issn.0578-1752.2020.12.008
    Abstract ( 317 )   HTML ( 10 )   PDF (449KB) ( 379 )   Save
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    【Background】 Descurainia sophia is the most widely distributed and harmful broad-leaved weed in winter wheat field, and florasulam is the most widely used ALS inhibitor herbicide for controlling broad-leaved weeds. After several years of application, the control effect of florasulam on D. sophia has been found to decline in some wheat fields, which may be related to the resistance. 【Objective】 The objective of this study is to clarify the resistance level and mechanism of D. sophia to florasulam, and to provide theoretical basis for the establishment of precise regional control of broad-leaved weeds in wheat field. 【Method】 A total of 40 populations of D. sophia were collected from winter wheat fields. Whole-plant dose response experiments were conducted to determine the resistance level of 40 populations to florasulam, tribenuron-methyl and MCPA in the greenhouse. At the same time, according to the ALS gene sequence of D. sophia, the genomic DNA of a single plant with high resistance to florasulam was extracted. The ALS gene sequences were obtained and compared with that of sensitive type Arabidopsis thaliana to find out the mutation site and the resistance mechanism. 【Result】 The results of resistance level determination showed that 32 of the 40 D. sophia populations were sensitive to florasulam, accounting for 80.00%. There were 3, 3 and 2 populations of low resistance, medium resistance and high resistance, respectively. JN-1, JNI-2 and LY-2 belonged to low resistance populations. Three populations LC-3, LY-4 and YT-1 belonged to medium resistance with resistance index (RI) of 49.00, 26.44 and 21.09, respectively. BZ-1 and DZ-3 belonged to high resistance populations with RI of 52.00 and 194.00, respectively. The ALS sequence analysis showed that there were mutations from CCT (pro) to TCT (Ser) or CTT (Leu) in amino acid 197 of ALS gene in BZ-1, while TGG (Trp) to TTG (Leu) in amino acid 574 of ALS gene in DZ-3. In addition, 19 of the 40 D. sophia populations were resistant to tribenuron-methyl, accounting for 47.50%, among them, 11 populations were low resistance, 6 populations were medium resistance with RI of 38.05, 13.55, 11.54, 10.45, 11.50, 11.02, and DZ-3 and LY-4 belonged to high resistance populations with RI of 244.75 and 68.50, respectively. All 40 populations were sensitive to MCPA. 【Conclusion】 Among the 40 populations of D. sophia collected from wheat fields in Shandong Province, 20.00% of them are resistance to florasulam, and the substitutions of amino acids occur in different positions in the populations with high resistance to florasulam. The resistance of D. sophia to tribenuron-methyl is still very serious, but all the populations are not resistant to MCPA. In view of the resistance area of D. sophia, the alternative and mixed use of herbicides with multiple mechanisms should be promoted, which can not only delay and control the development of resistance in weeds, but also expand the weed control spectrum and reduce the use of herbicides.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Spatio-Temporal Variations of Soil Organic Matter in Paddy Soil and Its Driving Factors in China
    LI DongChu,HUANG Jing,MA ChangBao,XUE YanDong,GAO JuSheng,WANG BoRen,ZHANG YangZhu,LIU KaiLou,HAN TianFu,ZHANG HuiMin
    Scientia Agricultura Sinica. 2020, 53(12):  2410-2422.  doi:10.3864/j.issn.0578-1752.2020.12.009
    Abstract ( 602 )   HTML ( 30 )   PDF (642KB) ( 570 )   Save
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    【Objective】 Spatio-temporal variation characteristics of soil organic matter (SOM) in paddy fields were evaluated in China, which would provide support for the improving the quality of cultivated land, adjustment of planting structure and climate change. 【Method】 The characteristics of SOM change and its driving factors were analyzed based on 338 national long-term monitoring sites, which were carried out in different rice planting regions from 1988 to 2017. The effects of SOM on soil bulk density were also studied. 【Result】 The SOM content of the paddy field in China increased by 3.49 g·kg-1 in averaged in the past 30 years, which was increased at the rate of 0.09-0.12 g·kg-1 annually. The average increased rate of SOM content ranked from high to low were Middle of Yangtze River region (MYR), South of China (SC), Northeast of China (NE), Southwest of China (SW) and Lower of Yangtze River (LYR). The average SOM content of the paddy field was 32.4 g·kg-1 in China at present, which was highest in MYR, followed by SC, NE, SW and LYR. The SOM content were responded by climate, soil types, nitrogen (N) fertilizer input, and cropping system. There was a significantly negative correlation relationship between SOM content and annual mean temperature in SW region and high latitude regions (P<0.05), and it was a positive correlation relationship in East region and low latitudes region (P<0.05). The average SOM content in gleyic paddy soil was significantly higher than that in other types of paddy soil. N fertilizer input at 200-300 kg N·hm-2·a-1 was conducive to SOM accumulation. There was strong response between soil bulk density and SOM, plough layer depth and SOM significantly (P<0.01). 【Conclusion】 The SOM content of the paddy field in China showed an upward trend in the past 30 years. The average increased rate of SOM content annually showed an increasing trend from South to North of China. The average annual temperature, annual precipitation, paddy soil types and nitrogen fertilizer application were the main driving factors to the SOM content of paddy field in China.

    Effects of Different Nitrogen Application Rates on Soil Organic Nitrogen Components and Enzyme Activities in Farmland
    JIAO YaPeng,QI Peng,WANG XiaoJiao,WU Jun,YAO YiMing,CAI LiQun,ZHANG RenZhi
    Scientia Agricultura Sinica. 2020, 53(12):  2423-2434.  doi:10.3864/j.issn.0578-1752.2020.12.010
    Abstract ( 610 )   HTML ( 57 )   PDF (695KB) ( 625 )   Save
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    【Objective】 The changes in enzyme activity conversion and soil organic nitrogen components in different nitrogen application rates were studied, and the relationship between enzyme activity and organic nitrogen component was analyzed. It provided a reference for rationally formulating fertilization amount and fertilization plan in the dry farming area of the Loess Plateau. 【Method】 Based on the different nitrogen (N) application rates (0 (CK), 52.5 (N1), 105 (N2), 157.5 (N3), and 210 (N4) kg N·hm-2) in Mazichuan village, Lijiabao town, Dingxi city, Loess Plateau, a long-term positioning test was set up on a spring wheat field. Bremner’s method was used to determine the content of organic nitrogen in the 0-40 cm soil layer after harvest, and the activities of four nitrogen-related enzymes were also measured. 【Result】 The order of distribution of soil organic nitrogen components was: amino acid nitrogen>acidolyzable ammonia nitrogen>unknown-acidolyzable nitrogen>amino sugar nitrogen. With the increase of N application rate, soil organic carbon, total nitrogen, total acid nitrogen, amino acid nitrogen, acidolyzable ammonium, urease activity and protease activity increased first and then decreased. Except for total nitrogen, all other components reached the maximum value under N2, and the total nitrogen content reached the maximum value under N3. Different soil layers in the same treatment decreased with the increasing soil depths. The results of redundancy analysis indicated that total nitrogen content and protease activity were the key factors affecting the distribution and transformation of organic nitrogen components in the Loess Plateau of Longzhong. C:N ratio was negatively correlated with all organic nitrogen components, while protease, organic carbon and urease were positively correlated with amino acid nitrogen. 【Conclusion】 In general, N2 treatment had the highest nitrogen supply potential, and the total nitrogen was the key factor affecting the transformation of organic nitrogen components in spring wheat in this area. The changes of soil organic nitrogen composition under different nitrogen application rates were obvious, which changed nitrogen in the conversion enzyme activity.

    Spatial-Temporal Changes of Newly Cultivated Land in Northern China and Its Zoning Based on Driving Factors
    WANG MingLei,SHI WenJiao
    Scientia Agricultura Sinica. 2020, 53(12):  2435-2449.  doi:10.3864/j.issn.0578-1752.2020.12.011
    Abstract ( 401 )   HTML ( 22 )   PDF (5912KB) ( 447 )   Save
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    【Objective】 The spatial and temporal change pattern of newly cultivated land is affected by both climate change and human activities. It is important to clarify the spatial and temporal change pattern of newly cultivated land and its driving factors to ensure the sustainable development of cultivated land resources and agriculture. 【Method】 Based on the data of newly cultivated land in north China of 1980s-1990s, 1990s-2000s and 2000s-2010s, the spatial-temporal changes of newly cultivated land in north China were analyzed by using gravity model and regional statistics; Based on the climate data of the three times, we calculated the active accumulated temperature (≥10℃, AAT10) and the standardized precipitation evapotranspiration index (SPEI), and analyzed the driving effect of climatic factors on cultivated land reclamation; Based on the socio-economic statistics since the 1980s, the paper analyzed the promotion of human factors on cultivated land reclamation by combining spatial superposition model and regional statistical model; Based on the combination of climatic and human factors and the method of geographical subdivision, the new cultivated land in north China was subdivided. 【Result】 The center of gravity of new cultivated land in northern China has shifted from northeast to northwest; the most cultivated land reclamation existed in warm and dry climate, but the proportion of cultivated land reclamation occurring in warm and humid climate conditions was gradually increasing; the increase of agricultural population, the advancement of agricultural technology and the influence of national policies provided necessary foundation for the expansion of cultivated land resources in Northeast China and Xinjiang. Abundant cultivated land reserve resources and flat terrain conditions in the Xinjiang, Heilongjiang, Jilin and other regions provided a possibility for land reclamation; based on the climate and human activities factors, the northern China was divided into seven regions, which included the zone of grass to cropland due to climate in Northeast, the zone of forest and grass to cropland due to climate and human activities in north region of Northeast of China, the zone of paddy field to dry land due to climate and human activities in middle region of Northeast, the zone of forest and grass to cropland due to climate and human activities in the North, the zone of ecological restoration dues to human activities in the loess plateau, the zone of unused land to cropland due to human activities in the northwest region, and zone of the oasis agriculture due to human activities in Xinjiang. 【Conclusion】 Since the 1980s, the spatial and temporal patterns of the new cultivated land in northern China showed obvious differences. The warm and dry climate did not become the limiting factor of cultivated land reclamation, but the wetter climate promoted the cultivated land reclamation to some extent. The increase of agricultural population, the progress of agriculture and the influence of national policies played an important role in cultivated land reclamation.

    HORTICULTURE
    Effects of Wax Coating on Off-Flavor Compound Accumulation in the Pulp of Satsuma Mandarin
    ZOU YunQian,ZHANG Li,WU FangFang,XU RangWei,XU Juan,HU ShiQuan,XIE HePing,CHENG YunJiang
    Scientia Agricultura Sinica. 2020, 53(12):  2450-2459.  doi:10.3864/j.issn.0578-1752.2020.12.012
    Abstract ( 761 )   HTML ( 18 )   PDF (1223KB) ( 395 )   Save
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    【Background】 Satsuma mandarin (Citrus unshiu) is the most important loose-skinned citrus fruit for fresh consumption in China. After washing, coating, packaging and other postharvest commercial handlings, the fruits often undergo a rapid accumulation of off-flavor compounds in the pulp, which obviously influences the taste and flavor and further affects the consumer's choice as well as the shelf life and market competitiveness of the fruit. 【Objective】 In order to optimize its postharvest commercial handling, the influence of waxing treatment on Satsuma mandarin was investigated, so as to prolong its shelf life and increase its market competitiveness. 【Method】 ‘Praecox Tanaka’ Satsuma mandarin fruits were treated with a series of dilutions of commercial waxes. Then, the internal CO2 and O2 content, ethanol and acetaldehyde concentrations and morphological changes of surface wax were monitored by GC-MS. In addition, the sensory attributes, including gloss, off-flavor, flavor, juiciness, mastication, sweetness, tartness and overall quality, were analyzed to comprehensively evaluate the effects of different treatments on fruit’s postharvest quality. 【Result】 The result showed that undiluted commercial wax T1 (402D) was able to increase the gloss of fruit surface, however, which could be detected clearly with scanning electron microscopy. The fruit surface was fully covered with artificial wax, which contributed to lower O2 and higher CO2 content. The internal content of CO2 in undiluted 402D treated Satsuma mandarin fruit was increased by 7.1% when the fruits were stored for 3 weeks, and the O2 content decreased from 1.6% to 1.4%. Compared to the control group, the content of CO2 was increased by 1.0% in treated fruits. Waxing also led to a rapid accumulation of off-flavor compounds such as ethanol and acetaldehyde in the pulp, and dilution of the mother solution could increase O2 content and decrease CO2 content at the fruit core, as well as retard the accumulation rate of off-flavor compounds. It was further found dilution of 402D with water to a proportion of 1:1 could be rather effective, as we observed that the ethanol content was 1 773.8 mg?L-1 in 402D treated fruits and the number lower to 560.5 mg?L-1in 402D/H2O (1:1) treated fruits. Also, the sensory evaluation changed from "highlighted" to "brighter", and the overall evaluation score increased significantly in 402D/H2O (1:1) treated fruits. Besides, constant cold chain (4℃) after the coating treatment could effectively inhibit off-flavor compound accumulation and prolong the shelf life. 【Conclusion】 Coating treatment was the key reason for the rapid accumulation of off-flavor compounds in Satsuma mandarin fruit. When the fruit was treated with 402D/H2O (1:1) and then stored at 4℃, the accumulation of ethanol and acetaldehyde, two typical odorous compounds, could be significantly reduced, which helped to greatly improve the taste and extend the shelf life to four weeks.

    Cloning and Functional Analysis of CsWRKYIIcs Transcription Factors in Tea Plant
    XIAO LuoDan, TANG Lei, WANG WeiDong, GAO YueFang, HUANG YiFan, MENG Yang, YANG YaJun, XIAO Bin
    Scientia Agricultura Sinica. 2020, 53(12):  2460-2476.  doi:10.3864/j.issn.0578-1752.2020.12.013
    Abstract ( 396 )   HTML ( 15 )   PDF (6550KB) ( 286 )   Save
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    【Objective】 The objective of this study was to clone CsWRKYIIcs transcription factors from the tea plant (Camellia sinensis) ‘Shaancha No.1’ and analyze their sequence characteristics, to investigate the expression patterns in different tissues and under abiotic stresses, and to verify the transcription activity, thus providing a basis for further exploring the functions of tea plants under abiotic stresses. 【Method】 Specific primers were designed based on the annotated WRKY sequences released in tea genome database. RT-PCR was used to amplify the cDNA sequences of CsWRKYIIcs from the tea plant ‘Shaancha No.1’, the bioinformatical tools were carried out to analyze the sequence characteristics, and the real-time fluorescence quantification PCR (qRT-PCR) was employed to investigate the expression patterns. Y2H assay was applied to verify the transcription activity. 【Result】 Nine cDNA sequences of the CsWRKYIIcs were obtained with the open reading frame length of 561, 960, 936, 978, 897, 912, 720, 1008 and 969 bp, encoding 186, 319, 311, 325, 298, 303, 239, 239, 335 and 322 amino acids, respectively. Except for the CsWRKYIIc7 which lacked zinc finger sequences, each of all the other CsWRKYIIcs contained one conserved WRKY domain and a typical C2H2-type zinc finger motif. WRKYIIcs had similar conserved motifs in different species, and CsWRKYIIcs from the tea plant showed higher identity at the amino acids level with those from dicotyledonous Arabidopsis thaliana and Vitis vinifera. Furthermore, multiple cis-elements related to abiotic stresses were predicted in the promoter regions, implying that the CsWRKYIIcs might involve in response to various abiotic stresses. qRT-PCR test results suggested that the expression patterns of nine CsWRKYIIcs in different tissues were quite specific, with higher expression level in roots and flowers than that in stems and leaves. Meanwhile, the nine CsWRKYIIcs showed different expression patterns when induced by drought, ABA, high temperature and high salinity stress; the expression of CsWRKYIIc1 and CsWRKYIIc7 changed most significantly, which were consistent with the result of putative cis-elements. In addition, the Y2H assay results indicated that all the nine CsWRKYIIcs had transcriptional activation activity. 【Conclusion】 Nine CsWRKYIIcs transcription factors cloned from the tea plant were involved in response to ABA, drought, high temperature and high salinity stress, and they might play a regulatory role of transcriptional activators. CsWRKYIIc1 and CsWRKYIIc7 might be used as candidate genes for further research into the anti-adversity function of tea plants.

    FOOD SCIENCE AND ENGINEERING
    Isolation, Structural Characterization and Antioxidant Activity of Black Sesame Melanin
    LI Jie,JIA XuChao,ZHANG RuiFen,LIU Lei,CHI JianWei,HUANG Fei,DONG LiHong,ZHANG MingWei
    Scientia Agricultura Sinica. 2020, 53(12):  2477-2492.  doi:10.3864/j.issn.0578-1752.2020.12.014
    Abstract ( 585 )   HTML ( 19 )   PDF (722KB) ( 472 )   Save
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    【Objective】 In order to provide more knowledge about the chemical structure and biological activity of black sesame melanin, black sesame melanin extract was isolated, and the structure of obtained fractions was elucidated by multiple spectroscopic methods. The in vitro antioxidant activity of obtained fractions was also evaluated. 【Method】 Melanin was extracted from black sesame hull by the methods of alkali extraction and acid precipitation. Crude black sesame melanin extract was then isolated by HW-40C size exclusion column, and the yields, color value and melanin content of obtained fractions were determined. The structure of each fraction was studied by extensive spectroscopic methods, including UV-Vis, elemental analysis (EA), fourier transform infrared spectroscopy (FT-TR), nuclear magnetic resonance spectroscopy (1H and 13C), X-ray photoelectron spectroscopy (XPS), electron paramagnetic resonance (EPR), and X-ray diffraction (XRD). Four methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assay, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assay, were used to evaluate the in vitro antioxidant activity of each fraction of black sesame melanin. 【Result】 Black Fr1 and brown Fr2 were isolated from black sesame melanin extract, with the yields of 60% and 24%, respectively; the molecular weights of these two fractions were 38 800 Da and 6 000 Da, respectively, and the melanin content of two fractions was 782.16 mg SME·g -1 DW and 884.66 mg SME·g-1 DW, respectively. Elemental analysis showed that Fr1 was eumelanin and Fr2 might be allomelanin. The UV-visible and infrared spectrum of Fr1 and Fr2 demonstrated that there were functional groups such as benzene ring, -OH, -NH2, -COOH and nitrogen heterocycle in the structure of both fractions. The results of1H-NMR indicated that Fr1 possessed more aliphatic hydrogen and substituted aromatic ring compared with Fr2. 13C-NMR spectrum indicated that there were more aliphatic carbon and carbonyl and less aromatic carbon in Fr1 compared with Fr2. The results of XPS showed that the content of functional groups of two fractions was different, C1s spectra indicated that the ratio of C-C(H) and C=O group of Fr1 was higher than that of Fr2, but the ratio of C-OH/C-N and O-C=O was lower than that of Fr2. N1s spectra showed that the ratio of C-NH of Fr1 was higher than that of Fr2, the ratio of aromatic N was lower than that of Fr2, and there was no C-NH3+ in Fr1. O1s spectra indicated that the ratio of C-OH of Fr1 was higher than that of Fr2, but the ratio of C=O of Fr1 was slightly lower than that of Fr2, and there was no absorbed H2O in Fr1. The results of EPR showed that these two fractions demonstrated strong paramagnetic resonance properties, the g values of Fr1 and Fr2 were 2.0078 and 2.0085, respectively, and the ΔHpp of Fr1 and Fr2 were 0.7430 and 0.6950 mT, respectively. The results of X-ray diffraction showed that both fractions of black sesame melanin were amorphous compounds, and there was a planar stack structure in Fr1. The IC50 values of DPPH free radical scavenging of Fr1 and Fr2 were 83.00 and 54.00 μg·mL-1, respectively. The IC50 values of ABTS free radical scavenging of Fr1 and Fr2 were 53.00 and 30.00 μg·mL-1, respectively. The FRAP values of Fr1 and Fr2 were 1.05 and 1.62 mmol FeE·g-1 DW, respectively. The ORAC values of Fr1 and Fr2 were 3 141.80 and 4 143.76 μmol TE·g -1DW, respectively. 【Conclusion】 Above results indicated that Fr1 was the main fraction of black sesame melanin, and spectroscopic analysis indicated that Fr1 was eumelanin and Fr2 might be allomelanin. Functional groups, such as carbonyl, hydroxyl, amino, aromatic ring and nitrogen heterocycle, existed in both fractions, and the aromaticity of Fr2 was higher than that of Fr1. The DDPH and ABTS free radical scavenging ability, and FRAP and ORAC antioxidant capacity of Fr2 were higher than that of Fr1.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Estimating the Correction Coefficient of Days to 100 kg in Licha Black Pig and Its Intercross with Berkshire
    ZHENG Hao,TU JinMin,XIONG XiuPing,ZHANG ZeKai,LI LongYun,HUANG LiBin,HUANG ZhiYong,XIAO ShiJun,GUO YuanMei
    Scientia Agricultura Sinica. 2020, 53(12):  2493-2501.  doi:10.3864/j.issn.0578-1752.2020.12.015
    Abstract ( 560 )   HTML ( 25 )   PDF (1540KB) ( 350 )   Save
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    【Objective】 The aims of this study were to estimate the correction coefficients CF and A of days to 100 kg (D100) and to compare their correction powers to each other in Licha black pig and its intercross with Berkshire boars, so as to provide a reference of correction coefficients for estimating D100 in Licha black pig and other Chinese indigenous pig breeds. 【Method】 In this study, 554 Licha black pigs with pigs from its intercross with Berkshire boars, including 941 F1 pigs and 755 F2 pigs, were randomly selected, and their body weights and ages in days had been automatically collected everyday by Winland performance testing system during their fatting periods. The data quality control was carried out according to the criteria, such as age ≥60 days and body weight ≥25 kg at start testing. Only the data have been passed quality control, which were used to estimate CF and A. CF was the ratio of two slopes, namely, the numerator was the slope of the regression line of weight to age, and the denominator was the slope of the straight line through the end point of performance testing (age and body weight at the testing end) and the original point (both age and weight were 0). A was the intercept of the regression equation of body weight to age, which could be obtained directly by fitting a regression equation of body weight to age. After the correction coefficients were estimated, an analysis of variance was carried out to test whether gender and population had effects on them, and the estimated CF and A were substituted into their respective correction formulae to calculate D100 (D100CF and D100A). A t-test was used to test whether there was a significant difference between D100CF and D100A, and their correlation coefficient was also calculated. Then, the regression equation of body weight to age for each individual was established, and regressive D100 (D100reg) for each individual was estimated by its regression equation. The corrected accuracies of CF and A were compared to each other by the correlation coefficients of D100CF and D100A with D100reg, and the means and standard deviations of the differences of D100CF and D100A to D100reg. Finally, the correction coefficients estimated from other two populations were used to estimate D100 in a certain population, and the correlation coefficients of D100CF and D100A with D100reg were calculated to evaluate whether the correction coefficient estimated from one population could be used in other populations. 【Result】 Gender and population had significant effects on the correction coefficients, so it was necessary to estimate the correction coefficient for each gender in each population. CF of stags (gilts) in Licha black pig and the F1 and F2 of its intercross with Berkshire boars were 1.426 (1.346), 1.340 (1.389) and 1.372 (1.380), respectively, and A were 60.65 (54.15), 49.92 (57.00) and 54.25 (57.53), respectively. There was no significant difference between D100CF and D100A (P = 0.3071), and their correlation coefficient was 0.9998 (P <0.0001). The correlation coefficients of D100CF and D100A with D100reg were equal or greater than 0.9917 (P<0.0001), there was no significant (P>0.05) between the differences of D100CF and D100A to D100reg. The means of the differences were -0.1385 and -0.2741 days, and the standard deviations were 2.5532 and 2.5446 days, respectively. The correlation coefficients of D100reg with D100CF and D100A, which were adjusted using the correction coefficients estimated from other two populations, were equal or greater than 0.9793 (P < 0.0001). 【Conclusion】 The correction coefficients CF and A of D100 estimated in this study were quite different from their counterparts using in National Swine Industry Technology System. There was no significant difference between the correction powers of CF and A, and both CF and A had a very high correction power under an appropriate range of body weight at the end of performance testing. In the three populations, the correction coefficients from other two populations had a very high correction power to a certain population, which indicated that the correction coefficient of D100 from Licha black pig could be used as a reference to other Chinese indigenous pig breeds.

    Effects of Dietary Energy Levels on Metabolism and Serum Parameters of Steppe Red Cattle
    LIU JiWei,ZHANG XiangLun,LI Xu,WANG Lei,QIN LiHong,BAN ZhiBin,WU Jian,ZHANG GuoLiang,WAN FaChun
    Scientia Agricultura Sinica. 2020, 53(12):  2502-2511.  doi:10.3864/j.issn.0578-1752.2020.12.016
    Abstract ( 529 )   HTML ( 32 )   PDF (410KB) ( 460 )   Save
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    【Objective】 This study was carried out to investigate the effects of dietary energy levels on gas metabolism, nutrient digestion and serum parameter of Steppe Red cattle. 【Method】 A total of twelve healthy Steppe Red cattle with body weight of 365.08±2.76 kg were randomly allocated into three groups. Each group was subject to one of the following 3 diets: 5.65, 6.05, and 6.43 MJ·kg-1 of net energy for gain, defining as LE, ME and HE, respectively. Experimental period was 20 days with 18 days of adaptive phase and 2 days of testing phase, respectively. Equipment of macrofauna open-type with respiration and calorimetric function was used for respiratory calorimetry trial and digestive and metabolic trial during testing phase. Blood samples were collected from jugular vein of Steppe Red cattle to isolate serum, and serum biochemical parameters were detected. 【Result】 Data showed that methane output, methane output as a proportion of dry matter intake, oxygen consumption, and heat production in HE group were higher than those of other groups, while carbon dioxide output in HE group was higher than that of ME group (P<0.05), and all the indices above increased linearly with the increase of dietary energy (P<0.05). Analysis of energy metabolism parameters showed that digestible energy intake, metabolic energy intake, gross energy digestibility, gross energy metabolic rate, fecal energy output and urine energy output were not affected by dietary treatments (P>0.05). Methane energy output and methane energy output as a proportion of gross energy in HE group were higher than other groups (P<0.05), and the two indices increased linearly as dietary energy level increased (P<0.05). Nitrogen metabolism indices did not differ among groups (P>0.05). Apparent digestibility of dry matter, organic matter, ether extract, neutral detergent fiber and acid detergent fiber were not affected by dietary energy level (P>0.05), whereas dry matter and organic matter apparent digestibility increased linearly with the increase of dietary energy (P=0.059). HE group exhibited increased serum total triglyceride and decreased urea nitrogen (P<0.05), and both indices were linearly changed as dietary energy level increased (P<0.05). Other serum parameters were not affected (P>0.05). 【Conclusion】 Overall, increasing dietary energy level could enhance nutrient utilization, whereas energy would lose in the form of methane energy when dietary energy level was too high. In general, medium energy group achieved the best energy utilization, and the appropriate supply quantity of the digestible energy and protein for 350 kg Steppe Red cattle were 128.12 MJ·d-1 and 749.50 g·d-1, respectively; the metabolic energy and protein were 121.78 MJ·d-1 were 678.75 g·d-1, respectively; the net energy for gain was 55.96 MJ·d-1.

    The Potential Role of MicroRNAs and MicroRNA-Mediated Competing Endogenous Networks During the Developmental Process of Apis mellifera ligustica Worker’s Midgut
    DU Yu,FAN XiaoXue,JIANG HaiBin,WANG Jie,FAN YuanChan,ZHU ZhiWei,ZHOU DingDing,WAN JieQi,LU JiaXuan,XIONG CuiLing,ZHENG YanZhen,CHEN DaFu,GUO Rui
    Scientia Agricultura Sinica. 2020, 53(12):  2512-2526.  doi:10.3864/j.issn.0578-1752.2020.12.017
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    【Objective】 MicroRNA (miRNA) can lead to the inhibition or degradation of mRNA via target binding, thus performing negative regulation of gene expression. MiRNAs play a key role in regulating the growth, development, immunity and cellular activity of insects. This study aims to deeply investigate differentially expressed miRNAs (DEmiRNAs) and their regulatory networks, and to systematically parse differential expression pattern and competing endogenous RNA (ceRNA) mediated mechanism underlying the development of Apis mellifera ligustica worker’s midgut. 【Method】 Midguts of A. m. ligustica 7- and 10-day-old workers were sequenced using small RNA-seq technology. After quality control, the sequencing data were mapped to the genome of A. mellifera, followed by identification of known miRNAs via mapping the mapped tags to miRBase database. Expression levels of miRNAs were calculated and normalized using tags per million (TPM) algorithm. Significant DEmiRNAs were screened out following the criteria of |log2fold change|≥1 and P≤0.05. Prediction, GO database annotation and KEGG database annotation of target mRNAs were conducted using related software. Based on the target binding relationship and previous findings, DElncRNA/DEcircRNA-DEmiRNA-DEmRNA networks associated with 9 signaling pathways (AMPK, PI3K-Akt, Wnt, cAMP, Hippo, mTOR, Toll/Imd, TGF-beta and MAPK signaling pathways) were visualized using Cytoscape software. CeRNA regulatory network of miR-342-y was further constructed followed by KEGG pathway annotation of target mRNAs involved in the network. Stem-loop RT-qPCR was used to verify the reliability of our sequencing data and differential expression of miRNAs. 【Result】 A total of 112 significant DEmiRNAs were identified in Am7 vs Am10 comparison group, including 38 up-regulated miRNAs and 74 down-regulated miRNAs, which could respectively target 7 434 and 9 559 mRNAs. These target mRNAs could be annotated to 21 and 23 functional terms associated with biological process, 16 and 17 terms associated with cellular component, and 10 and 11 terms associated with molecular function. Additionally, the targets could be annotated to 83 and 86 pathways related to material metabolisms such as fructose and mannose metabolism, purine metabolism, glycine, serine and threonine metabolism; 10 and 10 pathways related to cellular immune such as endocytosis, ubiquitin mediated proteolysis and melanogenesis; 5 and 5 pathways related to humoral immune such as MAPK, Jak-STAT and NF-κB; 13 and 11 signaling pathways related to development such as Hippo, FoxO and Notch. Furthermore, ceRNA regulatory networks of aforementioned 9 signaling pathways were constructed and analyzed, the result showed the existence of complex relationship among DEmiRNAs, DElncRNAs, DEcircRNAs and DEmRNAs; DEmiRNAs were located in the center, while DElncRNAs, DEcircRNAs and DEmRNAs were around the network. Further investigation suggested that miR-342-y was significantly down-regulated in the developmental processes of both worker’s midgut and larval gut of A. m. ligustica; this miRNA could target 3 DEcircRNAs, 4 DElncRNAs and 327 mRNAs. Stem-loop RT-qPCR result indicated that the differential expression trend of 4 DEmiRNAs was in accordance of that in sequencing result, confirming the authenticity of the sequencing data and differential expression pattern of miRNAs. 【Conclusion】 DEmiRNAs are likely to affect the growth and development of A. m. ligustica worker’s midgut by participating in regulation of the expression of genes engaged in material and energy metabolism-associated pathways, signaling pathways such as Hippo and Wnt as well as cellular and humoral immune pathways; ceRNA regulatory networks mediated by several key DEmiRNAs including miR-182-x, miR-291-y, miR-342-y and ame-miR-6001-3p are likely to play pivotal regulatory parts during the developmental process of the midgut of A. m. ligustica.